C-di-GMP, or Bis-(3′-5′)-cyclic dimeric guanosine monophosphate (cyclic-di-GMP or c-di-GMP), is a second messenger found in bacteria for intracellular signal transduction. Accumulating evidence suggest that c-di-GMP regulates a wide variety of bacterial behaviors such as biofilm formation, motility and virulence expression. The critical role of c-di-GMP in biofilm formation and virulence expression indicated that c-di-GMP may play vital roles in the chronic and acute infection caused by some pathogenic bacteria. The cyclic-di-GMP is a widespread bacterial messenger molecule with potential application as therapeutic agent for treating bacterial infection. Potential applications of cyclic-di-GMP and its analogues as a therapeutic agent include: Inhibiting colonization and biofilm formation by S. aureus; inhibiting proliferation of human colon carcinoma cells; an immunomodulatory molecule and has potent effects on antibody production and for preventing biofilm formation or promoting biofilm dispersal in medical equipment and devices such as lung inhalers, heart stents, heart valves; enteral tubing and the like.
Given the increasingly evident correlation between c-di-GMP signaling and bacterial pathogenesis, there is strong interest in the microbiology community to identify and characterize the components of c-di-GMP signaling network. Meanwhile, the use of c-di-GMP directly as a therapeutic agent for treating bacterial infection is also being explored. It was reported that exogenous c-di-GMP can reduce in vitro cell-cell interactions and biofilm formation of the gram-positive pathogen Staphylococcus aureus, including human methicillin-resistant S. aureus strains and animal clinical isolates. C-di-GMP was found to inhibit colonization and biofilm formation by S. aureus in a murine model of mastitis infection, resulting in the enhanced ability of the host to clear the pathogen. It was shown that c-di-GMP can modulate host cellular responses by inhibiting basal and growth factor-induced proliferation of human colon carcinoma cells. Studies also suggested that c-di-GMP is an immunomodulatory molecule with potent immunoprophylactic properties and vaccine adjuvant effects on antibody production.
Biochemical characterization of the macromolecules involved in c-di-GMP signaling, including the enzymes and receptors, requiring the use of c-di-GMP as substrate or ligand for biochemical assay. C-di-GMP can also be used as starting material for the synthesis of c-di-GMP analogues to be used as chemical probes or inhibitors. Although C-di-GMP could be obtained directly from bacterial cells, the low intracellular concentration of c-di-GMP makes this approach impractical for c-di-GMP production.
Cyclic di-GMP is synthesized by proteins with diguanylate cyclase activity. These proteins typically have a characteristic GGDEF motif, which refers to a conserved sequence of five amino acids. The GGDEF motif is housed within a GGDEF domain that has been observed in 681 bacteria species. Information on the domain can be found at the Pfam protein families database (http://pfam.sanger.ac.uk/family?acc=PF00990). Current enzymatic synthesis of c-di-GMP using known diguanylate cyclases (DGC) protein suffers from low production yield. Cyclic di-GMP is also an allosteric inhibitor of cellulose synthase in Gluconacetobacte. xylinus. C-di-GMP has been found to function in many bacteria as evidenced by the presence of large number of GGDEF, EAL and HD-GYP domain-containing proteins. GGDEF domains function as diguanylate cyclases (DGC) that catalyze the synthesis of c-di-GMP from GTP, whereas EAL and HD-GYP domains function as phosphodiesterases (PDE-A) for degrading c-di-GMP.
Currently, c-di-GMP is synthesized chemically or enzymatically in small quantity in research laboratories. The solution and solid-phase chemical synthesis approaches are expensive and time-consuming given the multi-step nature of the synthetic routes. On the other hand, the enzymatic production of c-di-GMP by using DGC domain-containing proteins only involves a single condensation of two GTP molecules. The enzymatic approach has been employed by several labs for the production of c-di-GMP for biochemical assays by using WspR, PleD or VCA0956, three mesophilic DGC domain-containing proteins from Pseudomonas aeruginosa, Caulobacter crescentus and Vibrio cholerae respectively. Previously, we found that the preparation of c-di-GMP by using mesophilic DGC proteins such as WspR only yielded limited amount of c-di-GMP.
DGC proteins are known to be tightly regulated by cellular c-di-GMP concentration. It is theorised that this regulation involves the I-site in PleD. The crystal structures of PleD and WspR have revealed that the I-site contains a RXXD motif.
There is intense current interest in c-di-GMP, and analogues, because of its role as a bacterial signalling molecule and its importance in bacterial virulence, pathogenesis, and biofilm formation. As a major focus of research and development there is a need to produce large volumes of c-di-GMP, or analogues. One of the major limitations of using well known DGC proteins such as WspR and PA290 for c-di-GMP production is the significant loss of enzymatic activity during c-di-GMP preparation. The currently available methods are not sufficient, producing only limited amounts of c-di-GMP. Access to large quantity of cyclic-di-GMP is prerequisite for the development of the molecule as an antibacterial agent. Previous methods used for synthesizing cyclic-di-GMP are too costly or time/labor consuming for this purpose.